CN107338310A - A kind of mark and application method for detecting wheat anti gibberellic disease gene PFT - Google Patents
A kind of mark and application method for detecting wheat anti gibberellic disease gene PFT Download PDFInfo
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Abstract
The invention discloses a kind of mark and application method for detecting or aiding in detection wheat anti gibberellic disease gene PFT.The present invention is by entering performing PCR amplification and sequencing to 204 parts of different cultivars PFT genes, it was found that in 3 kinds of allele of the gene, 14 nucleotide differences are shared between the allele of PFT I and the allele of PFT II, and the diagnostic mark PFT CAPS of PFT genes are developed according to the 1471st deoxyribonucleotide polymorphism of PFT genes.It is experimentally confirmed：Identifications and wheat anti gibberellic disease molecular mark of the molecular labeling PFT CAPS of the present invention available for wheat anti gibberellic disease gene PFT.
Description
Technical field
The invention belongs to biological technical field, and in particular to a kind of mark for detecting wheat anti gibberellic disease gene PFT and make
Use method.
Background technology
The wheat scab as caused by Fusarium graminearum etc. (Fusarium head blight, FHB) is a kind of stream extensively
Capable fungal disease, has a strong impact on yield and quality of wheat.China middle and lower reach of Yangtze River and the northeast area of wheat are its often hair, repeating transmission area
Domain.In recent years, popularization of straw-returning etc. influences under by Global climate change, wheat-Corn Rotation System system, the disease in expansion,
Exacerbation trend, it has also become Yellow River-Huai River region regular incidence does harm to.The current average annual occurring area of China's wheat scab is more than 533.3 ten thousand hm2,
Wherein 2012,2015 and 2016 especially serious.Such as the hm of 2012~2015 average annual occurring area of Jiangsu Province about 1,200,0002, more than this
Save wheat planting area 50%；Henan Province's generation in 2012 is the most serious, and area is 339.7 ten thousand hm2, take second place within 2016, be
174.0 ten thousand hm2.Susceptible seed contains mycotoxin, such as deoxynivalenol (Deoxynivalenol, DON), endangers people
Poultry health, have a strong impact on edible and feeding.
It is the economical and effective means for reducing head blight harm to cultivate disease-resistant variety.Scab resistance heredity is carried out both at home and abroad
Numerous studies, have positioned about 100 anti gibberellic disease QTL, are distributed in all chromosomes of wheat.Fhb1~Fhb7 etc. 7 is disease-resistant
Gene is by definite designation, wherein being located at the Fhb1 resistances of 3B the short arm of a chromosome most by force and stably from Sumai 3.In different heredity
Under background, Fhb1 effect slightly has difference, and head blight severity 20% or so can be averagely reduced under high Disease pressure.Fhb1 may be used also
DON is converted into the deoxynivalenol -3- glucosides (DON-3G) of low toxicity, mitigates toxin harm.
Fhb1 obtains relatively broad application in the North America spring wheat area that head blight is retransmitted, as North Dakota founds university's profit
Anti gibberellic disease hard red spring wheat kind Alsen, year 950000 hm of popularizing area are bred as with Sumai 32, account for the state wheat planting area
1/3 or so, mark detection contains Fhb1.University of Minnesota has been bred as anti gibberellic disease kind using Fhb1 molecular labelings auxiliary
Sabin, and most kinds (being) of the current seed selection in the school contain this gene.Japan cultivars Nobeokabouzu Komugi, Nyubai
Also contain Fhb1 with Shinchunaga etc., wherein Shinchunaga is used widely as the anti-source of head blight in Japan.
Scab resistance identification is affected by environment big, and molecular marker assisted selection is to cultivate the one preferred technique of disease-resistant variety.
Fhb1 has proved to be the most strong and stable anti gibberellic disease gene of resistance, the current limiting factor for utilizing marker assisted selection Fhb1
It is the diagnostic mark without low cost, though its linked marker Xgwm493, Xgwm533, UMN10 and Xsnp3BS-8 etc. are in breeding
In have certain application, but had a great influence by genetic background.Nearest Rawat etc. has cloned the gene, is PFT (pore-
Forming toxin-like) type, total length 3472bp, containing two extrons, an introne, encoding chimera agglutinin egg
In vain, important information is provided to develop its diagnostic mark.
The content of the invention
First purpose of the present invention is to provide a kind of primer for detecting or aiding in detection wheat anti gibberellic disease gene PFT
It is right.
Detection provided by the invention or auxiliary detection wheat anti gibberellic disease gene PFT primer pair are by primer 1 and primer 2 group
Into；
The primer 1 is the single strand dna shown in sequence 1；
The primer 2 is the single strand dna shown in sequence 2.
In above-mentioned primer pair, the mole ratio of the primer 1 and the primer 2 is 1:1.
Second object of the present invention is to provide detection or auxiliary detection wheat anti gibberellic disease gene PFT PCR reagent.
Detection provided by the invention or auxiliary detection wheat anti gibberellic disease gene PFT PCR reagent include above-mentioned primer pair.
In above-mentioned PCR reagent, the final concentration of the primer 1 and the primer 2 in the PCR reagent is 0.25 μ
mol/L。
Third object of the present invention is to provide a kind of reagent for detecting or aiding in detection wheat anti gibberellic disease gene PFT
Box.
Detection provided by the invention or auxiliary detection wheat anti gibberellic disease gene PFT kit include above-mentioned primer pair or
Above-mentioned PCR reagent.
Fourth object of the present invention is to provide above-mentioned primer pair or the new application of above-mentioned PCR reagent or mentioned reagent box.
The invention provides above-mentioned primer pair or above-mentioned PCR reagent or mentioned reagent box following 1) -4) in it is any in
Application：
1) detect or aid in detect wheat anti gibberellic disease gene PFT to be measured；
2) assist-breeding wheat-resistance to scab kind；
3) prepare detection or auxiliary detects wheat scab resistance gene PFT to be measured product；
4) product of seed selection wheat-resistance to scab kind is prepared.
The 5th purpose of the present invention is to provide a kind of side for detecting or aiding in detect wheat anti gibberellic disease gene PFT to be measured
Method.
The method that detection provided by the invention or auxiliary detect wheat anti gibberellic disease gene PFT to be measured comprises the following steps：
Enter performing PCR amplification to wheat to be measured with above-mentioned primer pair, obtain amplified production, PCR is expanded described in the digestions of Dra I
Product, obtain digestion products；Judge wheat anti gibberellic disease gene to be measured according to the pcr amplification product and the digestion products
PFT allele：
If wheat to be measured has a pcr amplification product, and pcr amplification product can not be by the digestions of Dra I, then wheat breed to be measured contains
There is allele PFT- I；
If wheat to be measured has a pcr amplification product, and pcr amplification product can be by the digestions of Dra I are 1567bp and 709bp two
Individual fragment, then wheat to be measured contain allele PFT- II；
If wheat to be measured contains allele PFT- III without pcr amplification product, wheat breed to be measured.
In the above method, the template of the PCR amplifications is the genomic DNA of wheat to be measured.
The 6th purpose of the present invention is to provide a kind of method of seed selection wheat-resistance to scab kind.
The method of seed selection wheat-resistance to scab kind provided by the invention is wheat product of the selection containing allele PFT- I
Kind carries out breeding.In actual applications, can be with PFT- containing allele I anti gibberellic disease parent with being free of allele equipotential base
The sense head blight parents of cause, above-mentioned primer pair and method choice PFT- containing allele I list is utilized in segregating generation
Strain, until selecting PFT- containing allele I homozygous lines.If Susceptible parent is the types of PFT- II, segregating generation PFT- I is pure
The band that individual plant digestion products only show that a size is 2277bp is closed, heterozygosis individual plant shows three bands, and size is respectively
2277bp, 1567bp and 709bp；If Susceptible parent is the types of PFT- III, then it is not required to carry out the digestions of Dra I, it is only necessary to above-mentioned primer
And heterozygosis individual plant homozygous to amplification segregating generation each individual plant DNA, PFT- I shows the band that a size is 2277bp, is free of
The individual plants of PFT- I are without amplified production.
In above-mentioned application or the above method, the wheat anti gibberellic disease gene PFT is existed only on wheat 3B chromosomes, and one
Individual copy, share following three kinds of allele：Allele PFT- I, allele PFT- II and allele PFT- III.It is described
Allele PFT- I sequence is as shown in sequence 3；The sequence of the allele PFT- II is as shown in sequence 4；The equipotential base
Because PFT- III is gene delection.
The present invention has found 3 kinds of equipotentials of the gene by the way that 204 parts of different cultivars PFT genes are entered with performing PCR amplification and sequencing
In gene, 14 nucleotide differences are shared between PFT- I and PFT- II, and according to the 1471st deoxyribose core of PFT genes
Thuja acid develops the diagnostic mark PFT-CAPS of PFT genes.It is experimentally confirmed：The molecular labeling PFT-CAPS of the present invention can
Identification and wheat anti gibberellic disease molecular mark for wheat anti gibberellic disease gene PFT.
Brief description of the drawings
Fig. 1 is PFT 3 kinds of allele.+ 1 is used as using the 1st base of initiation codon.
Fig. 2 is to PFT Gene Partial fragment amplification results with primer PFT-1F and PFT-2R.M：DL2000 DNA
marker；1~3 is the types of PFT- III；4 be the types of PFT- I；5 be the types of PFT- II.
Fig. 3 is the endonuclease recognition sequences of Dra I and cleavage site in the types of PFT- II.With the 1st base of initiation codon
It is used as+1.
Fig. 4 is the kind PFT-CAPS mark detections containing PFT- I or PFT- II.M：DL5000 DNA marker；1~4 is
The types of PFT- I；5~8 be the types of PFT- II.
Fig. 5 is the head blight disease index containing different PFT allele wheat breeds.
Embodiment
Experimental method used in following embodiments is conventional method unless otherwise specified.
Material used, reagent etc., unless otherwise specified, are commercially obtained in following embodiments.
Quantitative test in following embodiments, it is respectively provided with and repeats to test three times, results averaged.
The method of embodiment 1, molecular labeling PFT-CAPS acquisition and identification wheat anti gibberellic disease gene PFT
First, PFT 3 kinds of allele and the acquisition of polymorphic site
1st, the design of PFT specific amplifications primer
With Sumai 3 Fhb1 section (GenBank accession number：KX907434 PFT coding region sequences in) as probe,
EnsemblPlants databases (http://plants.ensembl.org/index.html) sequence alignment is carried out, obtain each
DNA homolog sequence, using gene order corresponding to the homologous sequence and Sumai 3 of acquisition as reference, design expands the gene open
The special primer of reading frame (ORF) 3B chromosomes, primer sequence are as shown in table 1.Primer binding sites are as shown in Figure 1.
Table 1, the special primer for expanding PFT gene opens reading frame (ORF)
2nd, PCR is expanded
Using the genomic DNA of 204 wheat breeds (variety name and source such as table 2) seedling leaves as template, using step
The primer of rapid 1 design enters performing PCR amplification, obtains pcr amplification product.PCR amplifications use hi-fi PCR enzyme KOD FX
(TOYOBO)。
3rd, the detection of pcr amplification product and clone
PCR primer is detected with 1.5% agarose gel electrophoresis, ethidium bromide (Ethidium bromide, EB) dyeing is purple
It is imaged under outer light.Gel reclaims DNA, converts competent escherichia coli cell after connecting carrier and cultivates, and each connection conversion is anti-
12 monoclonals of picking are answered, expands and reclaims direct Sequencing by Hua Da gene sequencing, or by target DNA fragments after cultivating.Sequence point
Analysis uses Geneious 10.0.7 (http://www.geneious.com) software.
As a result show：In 204 wheat breeds, PFT genes share 3 kinds of allele, are denoted as PFT- I, PFT- respectively
II and PFT- III.Wherein, the nucleotide sequence of the allele of PFT- I is as shown in sequence 3, and by with the allele of PFT- I
Wheat is named as the type wheats of PFT- I (totally 25 parts)；The nucleotide sequence of the allele of PFT- II will have as shown in sequence 4
The wheat for having the allele of PFT- II is named as the type wheats of PFT- II (totally 35 parts)；The allele of PFT- III is that PFT genes lack
Lose, and the wheat with the allele of PFT- III is named as the type wheats of PFT- III (totally 144 parts).Found by comparing：
14 nucleotide differences are shared between the allele of PFT- I and the allele of PFT- II, as shown in Figure 1.Wherein, 12 positioned at interior
Containing in son, respectively the 578th of PFT genes, the 620th, the 804th, the 998th, the 1023rd, the 1182nd,
1396, the 1471st, the 1480th, the 1493rd, (compared with PFT- I, PFT- II is shown in sequence 3 for 1571-1572 positions
PFT- I the 1571st and the 1572nd bit base between insert a base A) and 2128；2 are located in extron, respectively
For the 2255th and the 2270th, two SNP in extron belong to same sense mutation；The allele the 2128th of PFT- II
Position nucleotides (in introne) replaces with base A by bases G causes mRNA mistake montages, so that the gene loses function.
Table 2, Wheat Cultivars PFT genotype and head blight disease index
4th, scab resistance is identified
204 parts of wheat breeds in this research are tested for continuous 3 years in 2014-2016 in Hubei Prov. Acdemy of Agricutural Sciences South Lake
Field carries out head blight inoculated identification, and bacterial strain is that (in document, " head blight of Hubei Wheat kind (being) resists the bacterial strain in Huang gang 1
Property analysis " disclosed in cross).Experiment uses randomized complete-block design, two rows of plantation per material, row long 1m, line-spacing 0.25m, if
Repeat twice.Experiment terminates to be humidified using microcomputer timing atomizing device from heading stage to investigation, promotes morbidity.Chosen per cell
10 tassels bloomed, it is 1 × 10 with concentration5Individual mL-1Conidial suspension carries out spray inoculation.20d is adjusted after inoculation
Look into morbidity spike number, per fringe spikelet number and sick spikelet number, calculate disease index (the FHB index=incidences of disease × severity), wherein
The incidence of disease is the ratio of morbidity spike number and total spike number, and severity is the average value with spikelet number ratio per fringe disease spikelet number, with
Percentage.
Qualification result is as shown in table 2, and the average head blight disease index of 25 parts of type wheat breeds of PFT- I is 51.40%；
179 parts of PFT- II and the type wheat breed average out to 62.04% of PFT- III, difference is extremely significantly (Fig. 5).The type wheat product of PFT- I
Kind head blight disease index is lower by 17.15% than other two types wheat breeds.
2nd, molecular labeling PFT-CAPS acquisition and identification wheat anti gibberellic disease gene PFT method
1st, molecular labeling PFT-CAPS acquisition
Molecular labeling PFT-CAPS, molecular labeling are devised according to the 1471st of PFT genes the deoxyribonucleotide
PFT-CAPS sequence is as follows：
PFT-1F：ACAGGCACACACGGCTATAAATACC (sequence 1)；
PFT-2R：AGAACTGGATAGCACGCAAGCATAT (sequence 2).
2nd, PCR is expanded
Using the genomic DNA of 204 wheat breeds as template, performing PCR amplification is entered using primer PFT-1F and PFT-2R, point
Pcr amplification product is not obtained.
Amplification program is 95 DEG C of pre-degeneration 3min；94 DEG C of denaturation 30s, 65 DEG C of annealing 30s, 68 DEG C of extension 2min30s, totally 33
Individual circulation；Last 68 DEG C of extensions 7min, 4 DEG C of preservations.PCR reaction systems are as shown in table 3.
Table 3, PFT-CAPS mark PCR reaction systems
Enter row agarose gel electrophoresis electrophoresis to PCR primer and it is sequenced.As a result show：Containing the wheat product of PFT- I
Kind and the wheat breed containing PFT- II expand the fragment to 2277bp and 2276bp respectively, and are produced containing the wheat breeds of PFT- III without amplification
Thing (Fig. 2).
3rd, digestion
With the restriction endonucleases of Dra I respectively to being carried out containing the wheat breeds of PFT- I with the wheat breed PCR primer containing PFT- II at 37 DEG C
Digestion overnight.Endonuclease reaction system is as follows：PCR primer 3 μ L, 10 × Buffer 1.5 μ L, Dra I 0.8 μ L, ddH2The μ L of O 9.7,
The μ L of total reaction volume 15.
Digestion products are carried out with electrophoresis detection and it is sequenced.As a result show：The PCR productions of the wheat breed containing PFT- II
Thing can obtain two fragments that size is 1567bp and 709bp after the digestions of Dra I, and the wheat breed containing PFT- I can not be digested
(Fig. 3 and Fig. 4).
Therefore, the anti gibberellic disease gene PFT of wheat breed to be measured can be judged according to following method：With primer PFT-1F and
PFT-2R expands Wheat volatiles DNA to be measured, obtains amplified production, with the digestion amplified productions of Dra I, obtains digestion products, according to
Amplified production and digestion products judge the anti gibberellic disease gene PFT of wheat breed to be measured：
If wheat breed to be measured has an amplified production, and amplified production can not be by the digestions of Dra I, then wheat breed to be measured contains
Allele PFT- I；
If wheat breed to be measured has amplified production, and two that amplified production can be by the digestions of Dra I for 1567bp and 709bp
Fragment, then wheat breed to be measured contain allele PFT- II；
If wheat breed to be measured contains allele PFT- III without amplified production, wheat breed to be measured.
Claims (9)
1. a kind of primer pair for detecting or aiding in detection wheat anti gibberellic disease gene PFT, is made up of primer 1 and primer 2；
The primer 1 is the single strand dna shown in sequence 1；
The primer 2 is the single strand dna shown in sequence 2.
2. primer pair according to claim 1, it is characterised in that：The mole ratio of the primer 1 and the primer 2 is 1:
1。
3. a kind of PCR reagent for detecting or aiding in detection wheat anti gibberellic disease gene PFT, including drawing described in claim 1 or 2
Thing pair.
4. PCR reagent according to claim 3, it is characterised in that：The primer 1 and the primer 2 are in the PCR reagent
In final concentration be 0.25 μm of ol/L.
5. a kind of kit for detecting or aiding in detection wheat anti gibberellic disease gene PFT, including drawing described in claim 1 or 2
PCR reagent described in thing pair or claim 3 or 4.
6. the examination described in the PCR reagent or claim 5 described in primer pair or claim 3 or 4 described in claim 1 or 2
Agent box is following 1) -4) in it is any in application：
1) detect or aid in detection wheat scab resistance gene PFT；
2) assist-breeding wheat-resistance to scab kind；
3) detection or auxiliary detection wheat scab resistance gene PFT product are prepared；
4) product of assist-breeding wheat-resistance to scab kind is prepared.
7. a kind of detect or aid in the method for detecting wheat scab resistance gene PFT to be measured, comprise the following steps：
Enter performing PCR amplification to wheat to be measured with the primer pair described in claim 1, PCR primer is obtained, described in the digestions of Dra I
PCR primer, obtain digestion products；Judge wheat scab resistance gene to be measured according to the PCR primer and the digestion products
PFT：
If wheat to be measured has a pcr amplification product, and pcr amplification product can not be by the digestions of Dra I, then wheat breed to be measured contains
Position gene PFT- I；
If wheat to be measured has pcr amplification product, and two pieces that pcr amplification product can be by the digestions of Dra I for 1567bp and 709bp
Section, then wheat to be measured contains allele PFT- II；
If wheat to be measured contains allele PFT- III without pcr amplification product, wheat breed to be measured.
8. according to the method for claim 7, it is characterised in that：The template of the PCR amplifications is the genome of wheat to be measured
DNA。
9. a kind of method of seed selection wheat-resistance to scab kind, it is that wheat breed of the selection containing allele PFT- I is educated
Kind.
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| CN108486278A (en) * | 2018-06-26 | 2018-09-04 | 江苏省农业科学院 | Primer pair and application for identifying peaceful wheat No. 9 and its derived varieties scab resistance |
| CN108624711A (en) * | 2018-06-26 | 2018-10-09 | 江苏省农业科学院 | Primer and application of a pair for identifying peaceful wheat No. 9 and its derived varieties scab resistance |
| CN109055594A (en) * | 2018-09-05 | 2018-12-21 | 中国农业科学院作物科学研究所 | A kind of molecular labeling and application method for 895 anti gibberellic disease QTL of wheat in detecting |
| CN109207630A (en) * | 2018-11-12 | 2019-01-15 | 湖北省农业科学院粮食作物研究所 | It is a kind of for detecting the molecular labeling and application method of anti gibberellic disease QTL Qfhb.hbaas-1AS |
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| CN108486278A (en) * | 2018-06-26 | 2018-09-04 | 江苏省农业科学院 | Primer pair and application for identifying peaceful wheat No. 9 and its derived varieties scab resistance |
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| CN108486278B (en) * | 2018-06-26 | 2020-12-01 | 江苏省农业科学院 | Primer pair for identifying gibberellic disease resistance of Ningmai No. 9 and derivative variety thereof and application |
| CN109055594A (en) * | 2018-09-05 | 2018-12-21 | 中国农业科学院作物科学研究所 | A kind of molecular labeling and application method for 895 anti gibberellic disease QTL of wheat in detecting |
| CN109055594B (en) * | 2018-09-05 | 2021-11-16 | 中国农业科学院作物科学研究所 | Molecular marker for detecting gibberellic disease resistance QTL of Zhongmai 895 and using method |
| CN109207630A (en) * | 2018-11-12 | 2019-01-15 | 湖北省农业科学院粮食作物研究所 | It is a kind of for detecting the molecular labeling and application method of anti gibberellic disease QTL Qfhb.hbaas-1AS |
| CN109402236A (en) * | 2018-11-12 | 2019-03-01 | 长江大学 | The detection method of blast resistant gene Pi-25 in a kind of rice breed |
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